There is numerous membrane events associated with fertilization: phospholipids metabolism, membrane fusion during the sperm acrosome reaction, during sperm-egg fusion, during cortical granule exocytosis, and changes in microvilli. Surprisingly, only phospholipase C activation (PLC)(which leads to production of inositol 1,4,5- trisphosphate and sn 1,2-DAG, calcium release and protein kinase C activation, has been widely studied. Over the 15 years, my lab has used specialized lipid analysis equipment and protocols developed with Avanti Polar Lipids to undertake a unique series of studies on other lipid signaling pathways. Our recent publication (selected over other articles by ASBMB Today) suggests roles for phospholipase D and autotaxin in fertilization in an NIH-recognized model system (Xenopus laevis). Our long term goal is obtain a survey of lipid signaling paths activated during fertilization and detail their role in the induction of fertilization events. We will inhibit autotaxin activity, quantify the levels of lysophosphatidic acid (measured by HPLC/ELSD or mass spectrometry) and examine the effect upon fertilization events in Xenopus. In the last two specific aims, we will examine phospholipase D (which catalyzes phosphatidic acid formation) during fertilization. We also provide extensive Preliminary Results demonstrating that increased phosphatidic acid (PA) production is involved in fertilization events such as the elevation of DAG, the activation of Src, PLC, and elevation of IP3 and intracellular calcium. We will use proven methods to quantify (Kd) the affinity between LPA, PA and Src. We will alter lipase D activity (by use of constitutively active or dominant negative PLD) or morpholinos against Xenopus phospholipase D message. These studies will make use of our expertise and specialized equipment in lipid signaling and Xenopus biology obtained over the past 20 years. PUBLIC HEALTH RELEVANCE: Although there are many lipid events during fertilization, they have not been well studied. Funding of this proposal will support the first study on the role of autotaxin and phospholipase D in fertilization. This work should provide insight into the mechanism of fertilization and may lead to identification of defects associated with human infertility.